Glutamate is the major fast excitatory neurotransmitter in most regions of the central nervous system (CNS), including the hypothalamus. A decreased level of glutamate activity can be found during the use of antiglutamate receptor drugs (including some drugs of abuse), selective degeneration of glutamatergic neurons or projections, and embryonic development. Observations from other laboratories revealed increased cholinergic functions in the CNS during each of these three conditions. Our recent experiments in hypothalamic neuronal cultures indicated that a chronic blockade of ionotropic glutamate receptors dramatically increases excitatory acetylcholine (ACh) synaptic activity and the number of cholinergic neurons. Data suggested that during a long-term decrease in glutamate transmission in the hypothalamus in vitro, ACh, which normally exhibits only weak activity in the hypothalamus, plays the role of the major excitatory neurotransmitter and supports the excitation/inhibition balance. We also hypothesized that an increase in excitatory ACh transmission represents a novel form of neuronal plasticity that regulates the activity and excitability in neurons during a decrease in glutamate excitation. However, the mechanisms of glutamate-dependent regulation of ACh transmission in the CNS have not been studied. They will be studied in the proposed research in hypothalamic neurons. First, using rat hypothalamic cultures, we will test the hypothesis that during decrease in glutamate transmission ACh and glutamate are co-released from the same synaptic terminals. Second, using hypothalamic cultures, we will test the hypothesis that the induction of cholinergic phenotype in neurons is regulated through a CREB-dependent signal transduction pathway. Third, we will test the prediction that a chronic blockade of glutamate NMDA receptors in rats in vivo increases cholinergic phenotypic properties in hypothalamic neurons. This will be studied using electrophysiology, Ca 2+ imaging, immunostaining, and molecular biology. This project addresses the fundamental mechanisms of neuronal plasticity and regulation of neuronal activity that can take place in neuronal circuits during a decrease in glutamate excitation. Data obtained here may have an important clinical relevance, given that glutamate receptor antagonists are used for chronic treatment of patients, and some glutamate receptor antagonists are drugs of abuse.